Computational study of β-N-acetylhexosaminidase from Talaromyces flavus, a glycosidase with high substrate flexibility.

Kulik N, Slámová K, Ettrich R, Křen V - BMC Bioinformatics (2015)

Bottom Line:
Despite of high sequence identity to previously reported Aspergillus oryzae and Penicilluim oxalicum β-N-acetylhexosaminidases, this enzyme tolerates significantly better substrate modification.Understanding of key structural features, prediction of effective mutants and potential substrate characteristics prior to their synthesis are of general interest.To access the reliability of predictions on basis of the reported model, all results were confronted with available experimental data that demonstrated the principal correctness of the predictions as well as the model.

Background: β-N-Acetylhexosaminidase (GH20) from the filamentous fungus Talaromyces flavus, previously identified as a prominent enzyme in the biosynthesis of modified glycosides, lacks a high resolution three-dimensional structure so far. Despite of high sequence identity to previously reported Aspergillus oryzae and Penicilluim oxalicum β-N-acetylhexosaminidases, this enzyme tolerates significantly better substrate modification. Understanding of key structural features, prediction of effective mutants and potential substrate characteristics prior to their synthesis are of general interest.

Results: Computational methods including homology modeling and molecular dynamics simulations were applied to shad light on the structure-activity relationship in the enzyme. Primary sequence analysis revealed some variable regions able to influence difference in substrate affinity of hexosaminidases. Moreover, docking in combination with consequent molecular dynamics simulations of C-6 modified glycosides enabled us to identify the structural features required for accommodation and processing of these bulky substrates in the active site of hexosaminidase from T. flavus. To access the reliability of predictions on basis of the reported model, all results were confronted with available experimental data that demonstrated the principal correctness of the predictions as well as the model.

Conclusions: The main variable regions in β-N-acetylhexosaminidases determining difference in modified substrate affinity are located close to the active site entrance and engage two loops. Differences in primary sequence and the spatial arrangement of these loops and their interplay with active site amino acids, reflected by interaction energies and dynamics, account for the different catalytic activity and substrate specificity of the various fungal and bacterial β-N-acetylhexosaminidases.

Fig7: Substrate dynamics in the active site of TfHex. A. Active site with docked natural substrate chitobiose 1 after 10 ns of molecular dynamics simulation. B. Overlay of the active sites of TfHex with docked GlcNAc (4) in the beginning of molecular dynamics (grey) and during stable period (vivid color). Tyr 470, which normally fixes the substrate’s acetamido-group by hydrogen bond with its oxygen, established new interaction with oxygen at C1-atom. C. Overlay of pNP-GalNAc (3, grey) and pNP-GlcNAc (2, vivid) in the active site of TfHex after 10 ns molecular dynamics. Common residues are in red circles, Arg 218 with 3 is not shown. D. Overlay of the active sites of TfHex with docked pNP-GlcNAc-uronate (5; vivid color, yellow color - hydrogen bonds) and pNP-GlcNAc (2; grey).

Mentions:
The least favorable binding energy obtained with TfHex was observed when docking the product of hydrolysis of chitobiose and pNP-GlcNAc – N-acetylglucosamine (GlcNAc, 4). Here, the initial docking energy got less favorable by more than 1 kcal/mol during the molecular dynamics simulation. The position of GlcNAc in the active sites of both bacterial and fungal enzymes changed significantly during molecular dynamics, that was accompanied by changes in the hydrogen bonding interactions with the catalytic residues when compared to the natural substrate (Figure 7A-B), so that the position of the catalytic residues after simulations with GlcNAc facilitates the release of the product out of the active site (Additional file 1: Figure S5). The value of the calculated binding energy for the product can be used as a threshold for estimation of successful binding of the substrates, as it is generally accepted that the product should be quickly released from the active site. Moreover, we assume that the behavior of GlcNAc-hexosaminidase complexes during the equilibrated period of the simulation, which is characterized by stable root mean square deviation of C-alpha atoms and interaction energies, can predict the changes occurring in the active site before the departure of the product. In the recently published paper on insect hexosaminidase from O. furnacalis [25,37], the ‘open-close’ conformation of the active site during hydrolysis caused by the rotation of catalytic Gly 368 and Trp 448 was proposed. Based on the herein reported molecular dynamics simulations of fungal and bacterial β-N-acetylhexosaminidases we can enhance this view by proposing an additional set of changes regulating the product release: rotation of catalytic Glu side chain and shift of Cα-atoms of the catalytic residues, which could regulate the access to the active site.Figure 7

Fig7: Substrate dynamics in the active site of TfHex. A. Active site with docked natural substrate chitobiose 1 after 10 ns of molecular dynamics simulation. B. Overlay of the active sites of TfHex with docked GlcNAc (4) in the beginning of molecular dynamics (grey) and during stable period (vivid color). Tyr 470, which normally fixes the substrate’s acetamido-group by hydrogen bond with its oxygen, established new interaction with oxygen at C1-atom. C. Overlay of pNP-GalNAc (3, grey) and pNP-GlcNAc (2, vivid) in the active site of TfHex after 10 ns molecular dynamics. Common residues are in red circles, Arg 218 with 3 is not shown. D. Overlay of the active sites of TfHex with docked pNP-GlcNAc-uronate (5; vivid color, yellow color - hydrogen bonds) and pNP-GlcNAc (2; grey).

Mentions:
The least favorable binding energy obtained with TfHex was observed when docking the product of hydrolysis of chitobiose and pNP-GlcNAc – N-acetylglucosamine (GlcNAc, 4). Here, the initial docking energy got less favorable by more than 1 kcal/mol during the molecular dynamics simulation. The position of GlcNAc in the active sites of both bacterial and fungal enzymes changed significantly during molecular dynamics, that was accompanied by changes in the hydrogen bonding interactions with the catalytic residues when compared to the natural substrate (Figure 7A-B), so that the position of the catalytic residues after simulations with GlcNAc facilitates the release of the product out of the active site (Additional file 1: Figure S5). The value of the calculated binding energy for the product can be used as a threshold for estimation of successful binding of the substrates, as it is generally accepted that the product should be quickly released from the active site. Moreover, we assume that the behavior of GlcNAc-hexosaminidase complexes during the equilibrated period of the simulation, which is characterized by stable root mean square deviation of C-alpha atoms and interaction energies, can predict the changes occurring in the active site before the departure of the product. In the recently published paper on insect hexosaminidase from O. furnacalis [25,37], the ‘open-close’ conformation of the active site during hydrolysis caused by the rotation of catalytic Gly 368 and Trp 448 was proposed. Based on the herein reported molecular dynamics simulations of fungal and bacterial β-N-acetylhexosaminidases we can enhance this view by proposing an additional set of changes regulating the product release: rotation of catalytic Glu side chain and shift of Cα-atoms of the catalytic residues, which could regulate the access to the active site.Figure 7

Bottom Line:
Despite of high sequence identity to previously reported Aspergillus oryzae and Penicilluim oxalicum β-N-acetylhexosaminidases, this enzyme tolerates significantly better substrate modification.Understanding of key structural features, prediction of effective mutants and potential substrate characteristics prior to their synthesis are of general interest.To access the reliability of predictions on basis of the reported model, all results were confronted with available experimental data that demonstrated the principal correctness of the predictions as well as the model.

Background: β-N-Acetylhexosaminidase (GH20) from the filamentous fungus Talaromyces flavus, previously identified as a prominent enzyme in the biosynthesis of modified glycosides, lacks a high resolution three-dimensional structure so far. Despite of high sequence identity to previously reported Aspergillus oryzae and Penicilluim oxalicum β-N-acetylhexosaminidases, this enzyme tolerates significantly better substrate modification. Understanding of key structural features, prediction of effective mutants and potential substrate characteristics prior to their synthesis are of general interest.

Results: Computational methods including homology modeling and molecular dynamics simulations were applied to shad light on the structure-activity relationship in the enzyme. Primary sequence analysis revealed some variable regions able to influence difference in substrate affinity of hexosaminidases. Moreover, docking in combination with consequent molecular dynamics simulations of C-6 modified glycosides enabled us to identify the structural features required for accommodation and processing of these bulky substrates in the active site of hexosaminidase from T. flavus. To access the reliability of predictions on basis of the reported model, all results were confronted with available experimental data that demonstrated the principal correctness of the predictions as well as the model.

Conclusions: The main variable regions in β-N-acetylhexosaminidases determining difference in modified substrate affinity are located close to the active site entrance and engage two loops. Differences in primary sequence and the spatial arrangement of these loops and their interplay with active site amino acids, reflected by interaction energies and dynamics, account for the different catalytic activity and substrate specificity of the various fungal and bacterial β-N-acetylhexosaminidases.